Mass spectrometry analysis of mutant polypeptides in biological samples

ABSTRACT

The invention relates to a method for determining the presence of at least one distinct polypeptide in a biological sample comprising contacting the biological sample with a hydrolyzing agent, wherein the hydrolyzing agent is capable of hydrolyzing the distinct polypeptide in a sequence-specific manner such that at least one distinct peptide having a predetermined peptide measured accurate mass would result if the at least one distinct polypeptide were present in the biological sample, to obtain a hydrolyzed sample; bringing the hydrolyzed sample in contact with a substrate comprising at least one immobilized binding partner, wherein the at least one immobilized binding partner is capable of specifically binding the distinct peptide; removing the hydrolyzed sample from the substrate in a manner such that the distinct peptide would remain bound to the immobilized binding partner; contacting the substrate with an elution solution, wherein the distinct peptide would dissociate from the immobilized binding partner into the elution solution; subjecting a portion of the elution solution to liquid chromatography to segregate a plurality of molecules in the portion of the elution solution to obtain sorted molecules; determining the measured accurate mass of at least one sorted molecule present in the elution solution; and determining the presence of the at least one distinct polypeptide in the biological sample when a measured accurate mass of at least one molecule is substantially equal to the predetermined peptide measured accurate mass.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/931,445, filed Feb. 1, 2011, which claims priority to U.S.provisional patent application Ser. No. 61/300,212 filed Feb. 1, 2010,the entire contents of both applications, which are incorporated hereinby reference.

FIELD OF THE INVENTION

The invention encompasses methods and apparatuses for the rapidproteomic analysis of complex biological samples. Specifically, theinvention provides for rapid fractionation of the biological samples andsubsequent simultaneous analysis of the fractionate for the presence orabsence of a large number of polypeptides in general and mutant peptidesin particular.

BACKGROUND OF THE INVENTION

Personalized medicine is the application of genomic and molecular datato better target the delivery of health care to specific patients,facilitate the discovery and clinical testing of new products, and helpdetermine a person's predisposition to a particular disease orcondition.

On a technical level, personalized medicine depends on theidentification and detection of proteins, genes and genetic variation(“biomarkers”) that play a role in a given disease. Rodland, ClinBiochem. 2004 July; 37(7):579-83. The presence or absence of certainbiomarkers is then correlated with the incidence of a particular diseaseor disease predisposition. However, currently available methods forbiomarker analysis are associated with long waiting periods, high costand numerous technical hurdles.

Massspectrometry (MS) is an important method for the characterization ofproteins in biological samples. MS involves ionizing chemical compoundsto generate charged molecules or molecule fragments and measurement oftheir mass or mass-to-charge ratios. In a typical MS procedure, a sampleis loaded onto the MS instrument, and undergoes vaporization. Thecomponents of the sample are ionized by one of a variety of methods(e.g., by impacting them with an electron beam), which results in theformation of positively charged particles (ions). The positive ions arethen accelerated by a magnetic field. The computation of themass-to-charge ratio of the particles is based on the details of motionof the ions as they transit through electromagnetic fields, anddetection of the ions.

The two primary methods for MS ionization are electrospray ionization(ESI) and matrix-assisted laser desorption/ionization (MALDI).Generally, however, proteins must be enzymatically digested into smallerpeptides using a protease prior to ionization and analysis. Theresulting peptides are introduced into the mass spectrometer andidentified by peptide mass fingerprinting or tandem mass spectrometry.Identification of certain peptides allows the technician to infer theexistence of particular proteins in an original sample.

Proteins of interest to biological researchers are usually part of avery complex mixture of other proteins and molecules that co-exist inthe biological medium (e.g., serum, blood, urine, tissue sample, mucous,saliva, stool, etc.). This presents two significant problems. First, thetwo ionization techniques used for large molecules (i.e., ESI and MALDI)only work well when the mixture contains roughly equal amounts ofconstituents. However, different proteins tend to be present in widelydiffering amounts in biological samples. If such a mixture is ionizedusing ESI or MALDI, the more abundant species have a tendency to “drown”or suppress signals from less abundant ones. The second problem is thatthe mass spectrum from a complex mixture is very difficult to interpretbecause of the overwhelming number of mixture proteins. This complexityis exacerbated by the usually necessary enzymatic digestion of a proteinprior to MS analysis. As such, biological media must generally belaboriously pre-processed before MS analysis can be performed.

Two pre-processing methods are usually used to fractionate proteins, ortheir peptide products from an enzymatic digestion, prior to MSanalysis: Two-dimensional (“2-D”) gel electrophoresis and highperformance liquid chromatography (“HPLC”).

Two-dimensional (“2-D”) gel electrophoresis separates a mixture ofproteins by two properties in two dimensions on 2D gels. See e.g.,Kossowska et al., Postepy Hig Med Dosw (Online). 2009 Nov. 12;63:549-63; Ma et al., Electrophoresis. 2009 August; 30(15):2591-9.

2-D electrophoresis begins with 1-D electrophoresis but then separatesthe molecules by a second property in direction 90 degrees from thefirst. The result is that the molecules are spread out across a 2-D gel.Because it is unlikely that two molecules will be similar in twodistinct properties, molecules are more effectively separated in 2-Delectrophoresis than in 1-D electrophoresis. However, 2-Delectrophoresis is a time (30 minutes to 12 hours per sample) and laborintensive process requiring complex as well as very expensive equipmentand highly trained and experienced technicians.

Fractionation of peptides after enzymatic digestion into multiplefractions by HPLC (e.g. by SCX) prior to MS analysis is also commonlyused. Yet all these fractions must be analyzed separately, greatlyincreasing time and effort spent on a single sample.

Apart from these fractionation methods prior to MS analysis, MS itselfis often combined with HPLC which is generally referred to as “LC-MS.”LC-MS is a powerful technique used for many applications which has veryhigh sensitivity and specificity.

The “bottom-up” approach to proteomics involves protease digestion anddenaturation followed by LC-MS with peptide mass fingerprinting; orLC-MS/MS (or “two-stage” or “tandem MS”) to derive the sequence ofindividual peptides. See e.g., Shi et al., Anal Chem. 2009, Nov. 19;Grimsrud et al., Plant Physiol. 2009 Nov. 18; Kesic et al.,Retrovirology. 2009 Nov. 17; 6:105; Kilpatrick et al., Anal Chem. 2009Oct. 15; 81(20):8610-6. Wu et al., Anal Biochem. 2009 Nov. 3; Hartwig etal., Arch Physiol Biochem. 2009 Nov. 4; Caron et al., Mol CellProteomics. 2007 July; 6(7):1115-22. Epub 2007 Mar. 20.

A tandem mass spectrometer is one capable of multiple rounds of massspectrometry, usually separated by some form of molecule fragmentation.Tandem mass spectrometry allow for a variety of experimental sequences.Normally, a tandem MS has at least two mass spectrometers in seriesconnected by a chamber that can break a molecule into pieces. A samplepeptide is sorted and weighed in the first mass spectrometer (MS1),broken into pieces in the collision cell, and a peptide piece or piecesagain sorted and weighed in the second mass spectrometer (MS2). Manycommercial mass spectrometers are designed to expedite the execution ofsequences as single reaction monitoring (SRM), multiple reactionmonitoring (MRM), and precursor ion scan. In SRM, the MS1 allows only asingle mass through and MS2 monitors for a single user defined fragmention. MRM allows for multiple user defined fragment ions. Unfortunately,SRM and MRM require complex and expensive instrumentation and computerequipment.

LC-MS/MS is most commonly used for protcomic analysis of complexsamples. Samples of complex biological fluids like human serum may berun through an LC-MS/MS system and result in over 1000 proteins beingidentified. However, to achieve such results, samples are generallyfirst separated on an SDS-PAGE gel or strong cation exchange (“SCX”)HPLC.

Recently, it has been reported that the MRM assay can be coupled with astrategy to enrich certain peptides: Stable Isotope Standards withCapture by Anti-Peptide Antibodies (“SISCAPA”). In the method,anti-peptide antibodies immobilized on 100 nanoliter nanoaffinitycolumns are used to enrich specific peptides along with spikedstable-isotope-labeled internal standards of the same sequence. Uponelution from the anti-peptide antibody supports, SRM/MRM tandem MS isused to quantify the peptides (natural and labeled). See Whiteaker etal., Mol Cell Proteomics. 2009 Oct. 20; Ahn et al., J Proteome Res. 2009September; 8(9):4216-24; Kuhn et al., Clin Chem. 2009 June; 55(6):108-17. Epub 2009 Apr. 16; Anderson et al., Mol Cell Proteomics. 2009May; 8(5):995-1005. Epub 2009 Feb. 4; Anderson et al., Mol CellProteomics. 2006 April; 5(4):573-88. Epub 2005 Dec. 6; Anderson et al.,J Proteome Res. 2004 March April; 3(2):235-44; US Pub. Nos. 20060154318,20070231909, 20080217254.

The inventor has developed a simplified method that can rapidlyfractionate and analyze a biological sample for the presence or absenceof a particular protein or peptide analytes by measured accurate massalone (i.e., using only M1) without the need to for complex andexpensive tandem MS or MS2 procedures and equipment.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a method for determining thepresence of at least one distinct polypeptide in a biological samplecomprising contacting the biological sample with a hydrolyzing agent,wherein the hydrolyzing agent is capable of hydrolyzing the distinctpolypeptide in a sequence-specific manner such that at least onedistinct peptide having a predetermined peptide measured accurate masswould result if the at least one distinct polypeptide were present inthe biological sample, to obtain a hydrolyzed sample; bringing thehydrolyzed sample in contact with a substrate comprising at least oneimmobilized binding partner, wherein the at least one immobilizedbinding partner is capable of specifically binding the distinct peptide;removing the hydrolyzed sample from the substrate in a manner such thatthe distinct peptide would remain bound to the immobilized bindingpartner, contacting the substrate with an elution solution, wherein thedistinct peptide would dissociate from the immobilized binding partnerinto the elution solution, to obtain an elution solution comprising aplurality of molecules; determining the measured accurate mass of atleast one sorted molecule present in the elution solution; anddetermining the presence of the at least one distinct polypeptide in thebiological sample when a measured accurate mass of at least one moleculeis substantially equal to the predetermined peptide measured accuratemass.

In a preferred embodiment, the portion of the elution solution furthercomprises at least one standard peptide, wherein the at least onestandard peptide has the same amino acid sequence as the at least onedistinct peptide but a different measured accurate mass (by way ofincorporation of certain isotopes, for example). In this embodiment, theat least one standard peptide and the at least one distinct peptideco-segregate during liquid chromatography, confirming the chemicalidentity of the at least one distinct peptide in addition toidentification by accurate mass.

In another aspect, the invention provides a method for determining thepresence of at least one distinct polypeptide in a biological sample.The method comprises the contacting the biological sample with ahydrolyzing agent, wherein the hydrolyzing agent is capable ofhydrolyzing the distinct polypeptide in a sequence-specific manner suchthat at least one distinct peptide having a predetermined peptidemeasured accurate mass would result if the at least one distinctpolypeptide were present in the biological sample, to obtain ahydrolyzed sample; bringing the hydrolyzed sample in contact with asubstrate comprising at least one immobilized binding partner, whereinthe at least one immobilized binding partner is capable of specificallybinding the distinct peptide; removing the hydrolyzed sample from thesubstrate in a manner such that the distinct peptide would remain boundto the immobilized binding partner, contacting the substrate with anelution solution, wherein the distinct peptide would dissociate from theimmobilized binding partner into the elution solution, to obtain anelution solution comprising a plurality of molecules; segregating theplurality of molecules by liquid chromatography to obtain sortedmolecules; determining the measured accurate mass of at least one sortedmolecule present in the elution solution; and Determining the presenceof the at least one distinct polypeptide in the biological sample when ameasured accurate mass of at least one sorted molecule is substantiallyequal to the predetermined peptide measured accurate mass.

In one embodiment of various aspects of the invention, at least onestandard peptide is added to the plurality of molecules, wherein the atleast one standard peptide has substantially the same amino acidsequence as the at least one distinct peptide but a different measuredaccurate mass. In some embodiments, the standard peptide comprises anisotope such as ¹⁵N, ¹³C, ¹⁸O, or ²H. In some embodiments, the at leastone standard peptide and the at least one distinct peptide co-segregateduring liquid chromatography.

In another embodiment, at least two reference peptides are added to theplurality of molecules, wherein the at least two reference peptides haveknown a known liquid chromatography elution order.

In another embodiment, the presence of the at least one distinctpolypeptide is confirmed by the co-segregation of the at least onestandard peptide and the at least one sorted molecule and by thedifference in measured accurate mass between the at least one standardpeptide and the at least one sorted molecule.

In various embodiments of the invention, the liquid chromatography isHPLC, is reverse phase HPLC, or is reverse phase C18 HPLC.

In further embodiments, the plurality of molecules are segregated duringliquid chromatography according to hydrophobicity, according to charge,or according to retention time. In some embodiment, the at least onestandard peptide and the at least one distinct peptide have asubstantially identical retention time.

In another embodiment the at least one distinct polypeptide is a mutantpolypeptide relative to a wild type polypeptide. In further embodiments,the difference between the mutant polypeptide and wild type polypeptideis the addition, deletion and/or substitution of about 1 to about 10amino acid residues, or is the addition, deletion and/or substitution ofa post-translational modification on at least one amino acid residue. Insome embodiments, the post-translational modification is selected fromthe group consisting of phosphorylation, acetylation, ubiquitination,and glycosylation. In some embodiments, the at least one distinctpeptide comprises a mutant signature.

In yet another embodiment, the biological sample is derived from saliva,mucous, tears, blood, serum, lymph fluids, buccal cells, circulatingtumor cells, mucosal cells, biopsy tissue, cerebrospinal fluid, semen,feces, plasma, urine, a suspension of cells, or a suspension of cellsand viruses. In further embodiments, the biological sample comprisesbetween about 5,000 to about 20,000 different polypeptides, or betweenabout 7,500 to about 15,000 different polypeptides, or up to about100,000 different polypeptides and isoforms thereof. In some embodimentof the various methods of the invention, the biological sample isdepleted of a protein selected from the group consisting of albumin,IgG, IgA, transferrin, haptoglobin, and anti-trypsin; or combinationsthereof, prior to step (a).

In further embodiments, the hydrolyzing agent is an enzyme, a chemical(e.g., cyanogen bromide, BNPS-Skatole or formic acid). In furtherembodiments, the hydrolyzing agent is selected from the group consistingof trypsin, Lysine-C endopeptidase (LysC), arginine-C endopeptidase(ArgC), Asp-N, glutamic acid, endopeptidase (GluC), chymotrypsin andcombinations thereof.

In yet further embodiments the substrate comprises a gel matrix orpolymer beads. The gel matrix and/or the polymer beads may comprise alinking moiety to couple the immobilized binding partner.

In another embodiment the at least one immobilized binding partnercomprises an antibody or peptide binding fragment thereof.

In another embodiment the at least one immobilized binding partnercomprises a protein that specifically binds to the distinct peptide, orcomprises an antibody or antibody fragment that binds a mutant signaturepeptide, or comprises an antibody or antibody fragment that binds a wildtype peptide.

In another embodiment, the at least one immobilized binding partner is 1to about 500 or 1 to at least 500 immobilized binding partners, whereineach immobilized binding partner specifically binds to a differentdistinct peptide.

In another embodiment, the determining measure accurate mass includesthe step of ionization of the at least one molecule by AtmosphericPressure Chemical lonisation (APCI), Chemical lonisation (CI), ElectronImpact (EI), Electrospray lonisation (ESI), Fast Atom Bombardment (FAB),Field Desorption/Field Ionisation (FD/FI), Matrix Assisted LaserDesorption lonisation (MALDI), or Thermospray Ionisation. In someembodiments, the ionization is Electrospray Ionisation (ESI).

In further embodiments, the determining the measured accurate mass isperformed using an instrument with a mass accuracy of 5 ppm or less, oran instrument with a mass accuracy of 1 ppm or less.

Another aspect of the invention relates to apparatuses capable ofperforming the aforementioned methods. In some embodiments, theapparatus for carrying out the method comprises a mass spectrometer orcomprises an LC-MS.

A further aspect of the invention relates to kits comprising reagentsfor performing the aforementioned methods.

BRIEF DESCRIPTION OF THE FIGURES

A more complete understanding of the present invention may be obtainedby reference to the accompanying drawings, when considered inconjunction with the subsequent detailed description. The embodimentsillustrated in the drawings are intended only to exemplify the inventionand should not be construed as limiting the invention to the illustratedembodiments, in which:

FIG. 1. Coelution of synthetic heavy isotope peptides and biologicalpeptides in Example 10. S=synthetic peptide, B=biological peptide.

FIG. 2. As FIG. 1, but shows also coelution of 1C13 and 2C13 peptides(peptide isotopes containing 1 and 2 C13 atoms, respectively).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Previously, biological mixtures having more than 2-3 proteins typicallyrequire the additional use of tandem MS (“MS/MS”) and/or MS2 basedprotein identification to achieve sufficient specificity ofidentification. See Clauser et al., Anal. Chem., 1999, 71 (14), pp2871-2882. However, the methods of the invention do not require the useof MS2-peptide fragmentation to arrive at a definitive result withrespect to the presence or absence of a plurality of mutant polypeptidesin a biological sample. By not requiring MS2 analysis (e.g., SRM or MRMtechniques), the methods disclosed herein can be carried out in any labset up with basic affinity chromatography and MS1 capability. Moreover,the methods of the invention are readily adaptable for massimplementation without the need for specialized equipment requiringhighly skilled and experienced technicians. For example, the disclosedtechniques are readily adaptable to existing LC-MS systems (e.g., ThermoFischer's Exactive® System) or miniaturization utilizing microfluidictechnology.

Prior to describing various embodiments of the current invention, thefollowing definitions are provided:

As used herein the term “peptide” or “polypeptide” refers to a polymerformed from the linking, in a defined order, of preferably, α-aminoacids. The link between one amino acid residue and the next is called anamide bond or a peptide bond. Proteins are polypeptide molecules (orhaving multiple polypeptide subunits). The distinction is that peptidesare preferably short and polypeptides/proteins are preferably longeramino acid chains. The term “protein” is intended to also encompassderivatized molecules (e.g., post-translational modified polypeptides)such as glycoproteins and lipoproteins as well as lower molecular weightpolypeptides. The invention aims to identify the presence of certainproteins or polypeptides in a biological sample by identifying thepresence of a peptide portion of the protein or polypeptide, by way ofthe peptide's predicted measured accurate mass.

The term “standard peptide” as used herein, refers to a peptide thatis: 1) recognized as equivalent to a peptide of interest in the digest,e.g., the mutant signature peptide, by an appropriate immobilizedbinding partner(s); 2) co-elutes through LC with a peptide of interestin the digest, e.g., the mutant signature peptide; and 3) differs fromthe peptide of interest in a manner that can be distinguished by a massspectrometer. Preferably, the standard peptide has the same amino acidsequence as the mutant signature peptide but is synthesized utilizingelemental isotopes. Preferably, those isotopes are ¹⁵N, ¹³C, ¹⁸O or ²H.One or more standard peptides may be added to the biological samplebefore or after treatment with hydrolyzing agent such that it co-eluteswith the peptide of interest into the elution solution. Alternatively,the standard can be added directly to the elution solution. Exemplarystandard peptides are described in U.S. Pub. No. 20060154318.

In a preferred embodiment of the invention, at least one standardpeptide is added to either the biological sample, hydrolyzed sampleand/or elution solution such that the standard peptides enter into theLC-MS device at the same time as the peptides being analyzed. In thisembodiment, the standard peptides will preferably have the same aminoacid sequence as the peptide of interest as well as a different yetknown, mass. Most preferably, the standard peptide and the peptide ofinterest will co-elute through LC and therefore, have substantially thesame retention time. If the technician determines the presence of twoco-LC-eluting peptides different only with respect to a mass they canconfirm the presence of the peptide of interest and therefore, thepolypeptide of interest in the biological sample.

The term “wild type polypeptide” refers to the phenotype of the typicalform of a polypeptide or protein as it most often occurs in natureand/or which is associated with normal polypeptide or proteinphysiologic function.

Generally, a “mutant polypeptide” differs from the wild type polypeptidewith respect to amino acid sequence variation and/or post-translationalmodification (i.e., the “mutant signature”). The segment of aminoacid(s) of a mutant polypeptide that contains such variability is alsoreferred to herein as a “mutant signature peptide.”

Mutant polypeptides may be the result of normal inter-individual geneticvariability and/or may be associated with aberrant polypeptide orprotein physiologic function.

A mutant polypeptide may be associated with the addition, deletionand/or substitution of one or more preferably, about 1 to about 10,preferably about 1 to about 5, or more preferably about 1 to about 3, orabout 1 to about 2 amino acid residues relative to their wild typecounterpart.

By “post-translational modification” is meant a change to a polypeptidethat occurs after that polypeptide is translated from mRNA by aribosome. Pos-translational modifications include but are not limited tothe addition or deletion of biochemical functional groups such asacetate, phosphate, various lipids and carbohydrates. Non-limitingexamples of specific post-translational modifications include:Acylation; deacetylation; methylation, alkylation; amidation;biotinylation; formylation; gamma-carboxylation; glutamylation;glycosylation; glycation; glycylation; hydroxylation; iodination;isoprenylation; lipoylation; prenylation; GPI anchor formation;myristoylation; farnesylation; geranylgeranylation; diphthamide;ADP-ribosylation; flavin attachment; oxidation; palmitoylation;pegylaiion; phosphatidylinositol attachment; phosphopantetheinylation;phosphorylation—usually of a serine, tyrosine, threonine or histidineresidue; polysialylation; pyroglutamate formation; arginylation,sulfation; ubiquitination, sumoylation; and selenoylation.

As used herein, the term “biological sample” refers to a readilyobtainable mixture of a plurality of polypeptides present in varyingconcentrations. Preferred biological samples have up to about 100,000different polypeptides (including isoforms thereof). Preferably thebiological samples include about 5,000 to about 20,000 differentpolypeptides. More preferably, biological samples have about 7,500 toabout 15,000 different polypeptides. Most preferably, biological sampleshave about 10,000 different polypeptides. Generally, such samples areenvironmental, industrial, veterinary or medical in origin. Thepreferred biological samples include but are not limited to saliva,mucous, tears, blood, serum, lymph/interstitial fluids, buccal cells,mucosal cells, biopsy tissue, cerebrospinal fluid, semen, feces, plasma,urine, a suspension of cells, or a suspension of cells and viruses. Themost preferred biological samples are mammalian, more preferably humanbiopsy, serum, and urine.

Where the biological sample is blood, serum or lymph/interstitial fluid,the invention envisages an optional step of depleting the biologicalsample of common and disproportionally over-represented backgroundproteins not suspected of being associated with mutant polypeptides.Such proteins include but are not limited to albumin, IgG, IgA,transferrin, haptoglobin, and anti-trypsin; or combinations thereof. Theskilled artisan will recognized that such a step is carried out by basicaffinity chromatography techniques. As used herein the term “depleted”or “depleting” means markedly lessening the concentration of aparticular species in a solution, e.g., by more than or about 50%; morethan or about 60%; more than or about 65%; more than or about 70%; morethan or about 75%; more than or about 80%; more than or about 85%; morethan or about 90%; more than or about 92%; more than or about 95%; morethan or about 97%; more than or about 98%; more than or about 99%.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different enzymes, including but notlimited to trypsin, Lysine-C endopeptidase (LysC), arginine-Cendopeptidase (ArgC), Asp-N, glutamic acid endopeptidase (GluC) andchymotrypsin, V8 protease and the like, as well as chemicals, such asbut not limited to cyanogen bromide, BNPS-Skatole and formic acid. Inthe subject invention one or a combination of hydrolyzing agents cleavepeptide bonds in a protein or polypeptide, in a sequence-specificmanner, generating a predictable collection of shorter peptides (a“digest”). A portion of the biological samples are contacted withhydrolyzing agent(s) to form a digest of the biological sample. Giventhat the amino acid sequences of certain polypeptides and proteins inbiological samples are often known and that the hydrolyzing agent(s)cuts in a sequence-specific manner, the shorter peptides in the digestare generally of a predicable amino acid sequence. Preferably, thetreatment of a polypeptide with a hydrolyzing agent results in thepolypeptides being split into peptides about 5 to about 50 acids inlength. If the polypeptide in a biological sample is a mutantpolypeptide, at least one of the resulting peptides in the digest willbe a mutant signature peptide.

The term “hydrolyzed sample” (also referred to as a “digest”) refers toa solution of peptides that is the result of a portion of the biologicalsample having been exposed to one or more hydrolyzing agents.

The term “mass spectrometer” means a device capable of detectingspecific molecular species and measuring their accurate masses. The termis meant to include any molecular detector into which a polypeptide orpeptide may be eluted for detection and/or characterization. In thepreferred MS procedure, a sample, e.g., the elution solution, is loadedonto the MS instrument, and undergoes vaporization. The components ofthe sample are ionized by one of a variety of methods (e.g., byimpacting them with an electron beam), which results in the formation ofpositively charged particles (ions). The positive ions are thenaccelerated by a magnetic field.

The computation of the mass-to-charge ratio of the particles is based onthe details of motion of the ions as they transit throughelectromagnetic fields, and detection of the ions. The preferred errorrate of a mass spectrometer of the invention is 3 ppm or less, and mostpreferably 1 ppm or less. The present invention is surprisinglyadvantageous as it allows for the identification polypeptides in generaland mutant polypeptides in particular in biological samples by way ofaccurate mass alone without the need to use tandem MS or MS2. Thisadvantage arises in part due to enrichment of the peptides by theimmobilized binding partners in the elution solution relative to thehydrolyzed sample or a biological sample which thereby reduces theoverall complexity of the elution solution relative to the hydrolyzedsample or biological sample.

As used herein the term, “accurate mass” refers to an experimentally ortheoretically determined mass of an ion that is used to determine anelemental formula. For ions containing combinations of the elements C,H, N, O, P, S, and the halogens, with mass less than 200 Unified AtomicMass Units, a measurement with about 5 ppm or preferably lessuncertainty is sufficient to uniquely determine the elementalcomposition of molecules, e.g., enriched peptides, in the elutionsolution.

As used herein the term, “predetermined peptide accurate mass” refers tothe experimentally determined or calculated accurate mass of a peptidewith a known amino acid sequence (along with any associatedpost-translational modifications). The accurate mass of any suchspecific amino acid sequence may be readily calculated by one of skillin the art.

The term “substrate” includes any solid support or phase upon which abinding partner may be immobilized. Preferred supports are those wellknown in the art of affinity chromatography for example but not limitedto polymeric and optionally magnetic beads, polystyrene, sepharose oragarose gel matrices, or nitrocellulose membranes or the like.Preferably, the substrate contains a linkage moiety for coupling to theimmobilized binding partner. Such coupling might be through anavidin/biotin linkage or linkage through protein A or protein G agarose,for example.

The term “immobilized binding partner” refers to any of a large numberof different molecules or aggregates which are linked to the substrate.

In the invention, a binding partner functions by binding to apolypeptide or peptide in order to enrich it prior to determining itsaccurate mass. Preferably, mutant signature peptides are bound andenriched. Proteins, polypeptides, peptides, nucleic acids(oligonucleotides and polynucleotides), antibodies, ligands,polysaccharides, microorganisms, receptors, antibiotics, and testcompounds (particularly those produced by combinatorial chemistry) mayeach be a binding partner. The preferred binding partner is an antibodyor peptide-binding fragment thereof which specifically yet reversiblybinds peptides and/or mutant signature peptides.

It should be understood that the substrate can contain many differentimmobilized binding partners having a different binding specificity fora different polypeptide, peptide or mutant signature peptide. As such,immobilized binding partners might be derived from monoclonal sources orpolyclonal sera. Preferably, the substrate has 2 to about 500 or more;or at least 500 immobilized binding partners each specifically bindingto a different distinct peptide. This allows the technician tosimultaneously process and analyze the biological sample for thepresence of a large number of polypeptides in a manner not feasible withmultiplex PCR and immunohistochemical assays, and with far more accuracythan ELISA techniques and immunohistochemical assays. See also U.S. Pat.Nos. 6,441,140; 6,982,318; 7,198,896; 7,259,022; 7,300,753; 7,344,714;U.S. Ser. No. 11/484,485.

In one embodiment, the collection of immobilized binding partners on asubstrate specifically recognizes one or more mutant signature peptidesbut does not recognize or cross react with wild type peptides.

In another embodiment, the collection of immobilized binding partners ona substrate binds to both mutant signature peptides and wild typepeptides, e.g., by binding an epitope adjacent to or independent of themutant signature.

The term “antibody” may be any of the classes of immunoglobulinmolecules of any species, or any molecules derived therefrom, (e.g.,ScFv) or any other specific binding partners constructed by variation ofa conserved molecular scaffold so as to specifically bind a polypeptidepeptide fragment. The term “anti-peptide antibody” may be any type ofantibody that binds a specific peptide, mutant signature peptide, orother fragment for the purposes of enrichment from a sample or processedsample.

The term “specifically binds” is commonly used and well understood bythe person of ordinary skill in the art with respect to the bindingpartners. An antibody that specifically binds to a target may also bereferred to as a target-specific antibody. For example, amutant-specific antibody specifically binds to the mutant polypeptideand does not bind to the wild-type polypeptide. Non-limitingmutant-specific antibodies include an EGFR E746-A750del-specificantibody that does not bind got wild-type EGFR (e.g., product 2085 soldby Cell Signaling Technology, Inc., Danvers, Mass.) and an EGFRL858R-specific antibody that does not bind to wild-type EGFR (e.g.,product 3197 sold by Cell Signaling Technology, Inc). Additionalnon-limiting mutant-specific antibodies include antibodies such as thePhospho-c-Abl (Tyr245)-specific antibody sold by Cell SignalingTechnology, Inc. (product 2868) that specifically binds to c-Abl whenphosphorylated at Tyr245 but does not bind to wild-type c-Abl (i.e.,does not bind non-phosphorylated at Tyr245) and the Acetyl-Histone H4(Lys12)-specific antibody sold by Cell Signaling Technology, Inc.(product 2591) that specifically binds to Histone H4 only when it isacetylated on Lys12 but does not bind wild-type Histone H4 (i.e., doesnot bind Histone H4 when H4 is not acetylated on Lys12).

The term “elution solution” refers to a solution that when brought intocontact with the immobilized binding partner and substrate, results inthe dissociation of the polypeptide or peptide and preferably the mutantsignature peptide from the immobilized binding partner into the elutionsolutions. Determining the salt, pH and ionic conditions necessary forsuch functionality is well within the ordinary skill in the art.

Preferably, the elution solution is far less complex than the biologicalsample of the hydrolyzed sample. It is preferably substantially enrichedfor peptides which were bound to the immobilized binding partnersrelative to the polypeptides and peptides of the hydrolyzed sample.Preferably, the elution solution has about 1 to about 5000, morepreferably about 10 to about 1009 different peptides. Most preferably,the elution solution is enriched for mutant signature peptides. Thesubstantial reduction in the complexity of the elution solution relativeto the hydrolyzed sample and biological sample allows for efficientsorting of the enriched peptides in the elution solution during theLC-MS.

The term “vaporizing a portion of the elution solution” means that aportion of the elution solution is preferably transferred to a massspectrometer for vaporization and ionization.

The term “ionizing” refers to atmospheric pressure chemical ionization(APCI), chemical ionization (CI), electron impact (EI), electrosprayionization (ESI), fast atom bombardment (FAB), field desorption/fieldionization (FD/FI), matrix assisted laser desorption ionization (MALDI),and thermospray ionization. The preferred method of ionization is ESI.

As used herein, “ionized molecule” refers to molecules in the elutionsolution that have become charged and are ready to move into theelectric fields that will direct them into the mass analyzer of a massspectrometer. Preferably, the ionized molecules include ionizedpolypeptides, peptides and/or mutant signature peptides present in theelution solution. Most preferably, the ionized molecules are mutantsignature peptides.

“Chromatography” is a chemical separation technique that involvespassing a mixture dissolved in a “mobile phase” through a stationaryphase, which separates the analyte to be measured from other moleculesin the mixture based on differential partitioning between the mobile andstationary phases.

“Liquid chromatography” or “LC” as used herein is a separation techniquein which the mobile phase is a liquid. Liquid chromatography can becarried out either in a column or a plane. Preferably, the LC methodsand equipment of the invention are coupled with a mass spectrometer.

Preferably, liquid chromatography uses relatively high pressure isreferred to as high performance liquid chromatography (“HPLC”). HPLC ispreferably used herein to separate, identify, and quantify compoundsbased on characteristics including but not limited to theiridiosyncratic polarities and interactions with the column's stationaryphase. Preferably, HPLC utilizes different types of stationary phase(typically, hydrophobic saturated carbon chains), a pump that moves themobile phase(s) and analyte, e.g., peptide, through the column, and adetector that provides a characteristic retention time for the analyte.The HPLC preferably also provides other characteristic information(e.g., UV/Vis spectroscopic data for analyte). Peptide retention timevaries depending on the strength of its interactions with the stationaryphase, the ratio/composition of solvent(s) used, and the flow rate ofthe mobile phase.

In one embodiment, the HPLC involves reverse phase HPLC (“RP HPLC”). InRP HPLC, compounds are preferably separated based on their hydrophobiccharacter. Preferably, peptides are be separated by running a lineargradient of an organic solvent. The preferred RP HPLC method utilizes aC18 column.

With respect to the analysis of the peptides herein, a run-to-runretention time accuracy (e.g. +/−2 minutes in a 45-minute gradient) isachievable with current generic liquid chromatography pumps.

Liquid chromatography-mass spectrometry (“LC-MS”) is a technique thatcombines the physical separation capabilities of liquid chromatography(or HPLC) with the mass analysis capabilities of mass spectrometry.

A portion of the elution solution may be directly transferred to a massspectrometer. Alternatively, a portion of the elution solution issubject to further manipulation e.g., to concentrate, to sort, or toorder the peptides and/or polypeptides contained therein.

In the most preferred embodiment, a portion of the elution solution isanalyzed using an LC-MS device. Preferably the LC device separates andorders the peptides (e.g., by retention time) present in the elutionsolution prior to the MS1 measurement of their accurate mass. In oneembodiment, the retention times of the peptides present in the elutionsolution are matched with the measured accurate mass of the peptides. Inanother embodiment, the relative position of peptide elution isadditionally aligned with a set of peptides the LC elution order and/ortime of which is known (i.e., “reference peptides”). Mechanisms fordirecting solutions from liquid chromatography to mass spectrometers maybe found for example in U.S. Pub. No. 20080217254.

In a preferred embodiment, the accurate mass of a molecule in theelution solution, e.g., a peptide combined with the LC co-elution ofstandard peptides of the same amino acid sequence but with a differentaccurate mass resulting from the incorporation of certain elementalisotopes. Preferably, the co-elution, e.g. on a massspectrometer-coupled C18 column serves as confirmation of chemicalidentity and makes the identification of the peptide in the elutionsolution sample unambiguous.

The invention relates to methods and apparatuses which allow atechnician to rapidly fractionate and analyze a complex biologicalsample for the presence or absence of a particular polypeptide bythrough affinity chromatography, liquid chromatography and MS1 massspectrometry.

Future improvements in accuracy of mass measurement may makechromatographic retention time of peptides unnecessary as a secondarycharacteristic. In that case, direct injection onto a mass spectrometerafter enrichment by IAP, without prior LC-separation, may become aviable option.

One aspect of the invention involves a method for determining thepresence or absence of a plurality of polypeptides in a biologicalsample such as biopsy, blood or serum. In a preferred embodiment of thisaspect of the invention, the polypeptides in the biological sample aredigested with a sequence-specific hydrolyzing agent such as theproteases GluC and/or trypsin. The resulting peptides of interest in thedigest are then enriched by immunoaffinity purification using a varietyof substrate-immobilized antibodies that recognize the peptides ofinterest. The affinity purified peptides of interest are then separatedand ordered using LC. Subsequently, their accurate mass is determined byMS. The combination of a peptide's measured accurate mass and itsposition in the order of peptides sorted by LC (preferably determined byretention time), allows the technician to determine the identity of thepeptide and determine whether it corresponds to the peptide of interest.Once the technician determines the presence of a particular peptide,they can extrapolate the presence of the polypeptide giving rise to thatpeptide in the biological sample.

In a preferred embodiment of this aspect of the invention, at least onestandard peptide is added to either the biological sample, hydrolyzedsample and/or elution solution such that the standard peptides enterinto the LC-MS device at the same time as the peptides being analyzed.In this embodiment, the standard peptides will preferably have the sameamino acid sequence as the peptide of interest as well as a known massdifferent from the peptide of interest, e.g, by way of incorporation ofcertain isotopes. Most preferably, the standard peptide and the peptideof interest will co-elute during LC and therefore, have substantiallythe same retention time. If the technician determines the presence oftwo co-LC-eluting peptides different only with respect to a mass theycan confirm the presence of the peptide of interest and therefore, thepolypeptide of interest in the biological sample.

Certain diseases result from the presence or over-expression of mutantpolypeptides. For example, Huntington's and Machado-Joseph disease arefatal inherited diseases caused by abnormal repeats of a small segmentin a person's DNA resulting in the body producing malfunctioningproteins that cause the diseases. Further examples of such disease areto Epidermolysis bullosa, sickle-cell disease, and SOD1 mediated ALS.

Additionally, an oncogene is a gene that, when mutated or expressed athigh levels, can turn a normal cell into a tumor cell. A proto-oncogeneis a normal gene that can become an oncogene due to mutations orincreased expression. Proto-oncogenes code for proteins that help toregulate cell growth and differentiation. Proto-oncogenes are ofteninvolved in signal transduction and execution of mitogenic signals,usually through their protein products. Upon activation, aproto-oncogene (or its product) becomes a tumor-inducing agent, anoncogene. Examples of proto-oncogenes include RAS, WNT, MYC, ERK, andTRK, epidermal growth factor receptor (EGFR), platelet-derived growthfactor receptor (PDGFR), and vascular endothelial growth factor receptor(VEGFR), HER2/neu, Src-family, Syk-ZAP-70 family, and BTK family oftyrosine kinases, the Abl gene in CML—Philadelphia chromosome, c-Sis andRaf kinase. In one non-limiting example, the proto-oncogene EGFR cangive rise to several oncogenic mutants, and different mutants may beassociated with different cancers. For example, many types of EGFRmutations have been reported, but the most common non-small cell lungcancer (NSCLC)-associated EGFR mutations are the 15-bp nucleotidein-frame deletion in exon 19 (E746-A750del) and the point mutationreplacing leucine with arginine at codon 858 in exon 21 (L858R) (Pao,W., et al., Proc Natl Acad Sci USA, 2004. 101(36): p. 13306-11; Riely,G. J., et al., Clin Cancer Res, 2006. 12(24): p. 7232-41; and Kosaka,T., et al., Cancer Res, 2004. 64(24): p. 8919-23). These two mutationsrepresent 85-90% of EGFR mutations in NSCLC patients. Importantly,patients with these mutations have been shown to respond well to EGFRinhibitors including Gefitinib and Erlotinib (Riely, G. J., et al., ClinCancer Res, 2006. 12(24): p. 7232-41; Inoue, A., et al., J Clin Oncol,2006. 24(21): p. 3340-6; Marchetti, A., et al., J Clin Oncol, 2005.23(4): p. 857-65; and Mitsudomi, T., et al., J Clin Oncol, 2005. 23(11):p. 2513-20). Therefore detection of these mutations is an importantmethod to improve treatment of lung cancer patients.

A tumor suppressor gene, or antioncogene, is a gene that protects a cellfrom one step on the path to cancer. When this gene is mutated to causea loss or reduction in its function, the cell can progress to cancer,usually in combination with other genetic changes. Non-limiting examplesof tumor suppressor genes are p53 tumor-suppressor protein encoded bythe TP53 gene, PTEN, APC and CD95.

Accordingly, another aspect of the invention is a method of determiningwhether a patient is predisposed to or has a disease associated with thepresence of a mutant polypeptide. Such mutant polypeptides may resultfrom mutations in any of the above-listed genes including oncogenes,proto-oncogenes and tumor suppressor genes. In the preferred embodimentof this aspect of the invention, the technician can determine whether apatient is predisposed to or has a disease associated with the presenceof a mutant polypeptide, if that mutant polypeptide is present in abiological sample obtained from the patient. Preferably, the patient'sbiological sample is simultaneously assayed for the presence of aplurality of mutant polypeptides according to the methods disclosedherein.

Another aspect of the invention relates to a method of determiningwhether a particular therapeutic regime or agent is effective inreducing the levels of a mutant polypeptide in a patient. In thepreferred embodiment of this aspect of the invention, the method is usedto determine the efficacy of a drug which is being provided to a patienthaving a particular disease, by determining whether the levels of mutantpolypeptide associated with the disease are reduced in the biologicalsample. The preferred therapeutic agents of this embodiment are smallmolecules, polypeptides or nucleic acids, e.g., antisense nucleic acids,siRNA/miRNA constructs, etc. used to knockdown the expression of genesassociated with the production of mutant polypeptides or directly causethe degradation of mutant polypeptides.

Another aspect of the invention relates to methods of screening drugcandidates for potential use as therapeutic agents for diseaseassociated with mutant polypeptides. In the preferred embodiment, acandidate drugs are given to a test animal. Preferably, the test animalhas a disease associated with a mutant polypeptide. Biological samplesfrom the animal are preferably assayed using the methods disclosedherein to determine if administration of the drug candidate has resultedin an increase or decrease in the level of the mutant polypeptide in thebiological sample. Preferably, the test animal's biological sample issimultaneously assayed for the presence of a plurality of mutantpolypeptides according to the methods disclosed herein.

A further aspect of the invention relates to methods of determiningwhether a particular test agent can give rise to a disease associatedwith the presence of one or more mutant polypeptides. In the preferredembodiment of this aspect of the invention, a normal or geneticallysensitized animal (e.g., one containing a single copy of a loss offunction mutation in a tumor suppressor gene) is provided with a testagent. Biological samples from the animal are preferably assayed usingthe methods disclosed herein to determine if administration of the testagent has resulted in an increase in the level of a mutant polypeptidein the biological sample. Preferably, the animal's biological sample issimultaneously assayed for the presence of a plurality of mutantpolypeptides according to the methods disclosed herein.

Another aspect of the invention relates using the methods disclosedherein to determine whether a patient is predisposed to or has a diseaseassociated with a mutant polypeptide. One embodiment of this aspect ofthe invention comprises: obtaining a biological sample from a patientsuspected of being predisposed to or having the disease; contacting thebiological sample with a hydrolyzing agent, wherein the hydrolyzingagent is capable of hydrolyzing the mutant polypeptide in asequence-specific manner such that at least one mutant signature peptidehaving a predetermined mutant signature peptide measured accurate masswould result if the at least one mutant polypeptide were present in thebiological sample, to obtain a hydrolyzed sample; bringing thehydrolyzed sample in contact with a substrate comprising at least oneimmobilized binding partner, wherein the at least one immobilizedbinding partner is capable of specifically binding the mutant signaturepeptide; removing the hydrolyzed sample from the substrate in a mannersuch that the mutant signature peptide would remain bound to theimmobilized binding partner; contacting the substrate with an elutionsolution, wherein the mutant peptide would dissociate from theimmobilized binding partner into the elution solution; determining themeasured accurate mass of at least one molecule in the elution solution;and determining that the patient is predisposed to or has the diseasewhen a measured accurate mass of the at least one molecule issubstantially equal to the predetermined mutant signature peptidemeasured accurate mass.

A further aspect of the invention relates to using the methods disclosedherein to determine whether a therapeutic agent is effective in treatinga patient having a disease associated with a mutant polypeptide. In apreferred embodiment of this aspect of the invention, the methodcomprises obtaining at least one control biological sample from thepatient prior to administration of the therapeutic agent; obtaining atleast one test biological sample from the patient after administrationof the therapeutic agent; contacting the control biological sample witha hydrolyzing agent, wherein the hydrolyzing agent is capable ofhydrolyzing the mutant polypeptide in a sequence-specific manner suchthat at least one mutant signature peptide having a predetermined mutantsignature peptide measured accurate mass would result if the at leastone mutant polypeptide were present in the control biological sample, toobtain a control hydrolyzed sample; contacting the test biologicalsample with the hydrolyzing agent, to obtain a test hydrolyzed sample;bringing the control hydrolyzed sample in contact with a controlsubstrate comprising at least one immobilized binding partner, whereinthe at least one immobilized binding partner is capable of specificallybinding the mutant signature peptide; bringing the test hydrolyzedsample in contact with a test substrate comprising the immobilizedbinding partner; removing the control hydrolyzed sample from the controlsubstrate in a manner such that the mutant signature peptide wouldremain bound to the immobilized binding partner, removing the testhydrolyzed sample from the test substrate in a manner such that themutant signature peptide would remain bound to the immobilized bindingpartner, contacting the control substrate with a control elutionsolution, wherein the mutant peptide would dissociate from theimmobilized binding partner into the control elution solution;contacting the test substrate with a test elution solution, wherein themutant peptide would dissociate from the immobilized binding partnerinto the test elution solution; determining the measured accurate massof at least one molecule in the control elution solution; determiningthe measured accurate mass of at least one molecule in the test elutionsolution; determining the presence of the mutant polypeptide in thecontrol biological sample when a measured accurate mass of at least onemolecule in the control elution solution is substantially equal to thepredetermined mutant signature peptide measured accurate mass;determining the presence of the mutant polypeptide in the testbiological sample by when a measured accurate mass of at least onemolecule in the test elution solution is substantially equal to thepredetermined mutant signature peptide measured accurate mass;determining the efficacy of the therapeutic agent when the mutantpolypeptide is present in the control biological sample in a greateramount than in the test biological sample.

Yet a further aspect of the invention relates to the use of the methodsdisclosed herein to determine whether a test agent can result in adisease in a test subject associated with mutant polypeptide. The testsubject may be for example an animal or human. In a preferred embodimentof this aspect of the invention, the method comprises obtaining at leastone control biological sample from the test subject prior toadministration of the agent; obtaining at least one test biologicalsample from the test subject after administration of the agent;contacting the control biological sample with a hydrolyzing agent,wherein the hydrolyzing agent is capable of hydrolyzing the mutantpolypeptide in a sequence-specific manner such that at least one mutantsignature peptide having a predetermined mutant signature peptidemeasured accurate mass would result if the at least one mutantpolypeptide were present in the control biological sample, to obtain acontrol hydrolyzed sample; contacting the test biological sample withthe hydrolyzing agent, to obtain a test hydrolyzed sample; bringing thecontrol hydrolyzed sample in contact with a control substrate comprisingat least one immobilized binding partner, wherein the at least oneimmobilized binding partner is capable of specifically binding themutant signature peptide; bringing the test hydrolyzed sample in contactwith a test substrate comprising the immobilized binding partner;removing the control hydrolyzed sample from the control substrate in amanner such that the mutant signature peptide would remain bound to theimmobilized binding partner; removing the test hydrolyzed sample fromthe test substrate in a manner such that the mutant signature peptidewould remain bound to the immobilized binding partner, contacting thecontrol substrate with a control elution solution, wherein the mutantpeptide would dissociate from the immobilized binding partner into thecontrol elution solution; contacting the test substrate with a testelution solution, wherein the mutant peptide would dissociate from theimmobilized binding partner into the test elution solution; determiningthe measured accurate mass of at least one molecule in the controlelution solution; determining the measured accurate mass of at least onemolecule in the test elution solution; determining the presence of themutant polypeptide in the control biological sample when a measuredaccurate mass of at least one molecule in the control elution solutionis substantially equal to the predetermined mutant signature peptidemeasured accurate mass; determining the presence of the mutantpolypeptide in the test biological sample when a measured accurate massof at least one molecule in the test elution solution is substantiallyequal to the predetermined mutant signature peptide measured accuratemass; determining that the test agent can result in a disease associatedwith a mutant polypeptide when the mutant polypeptide is present in thetest biological sample but not in the control biological sample.

In embodiments of this aspect of the invention involving human testsubjects, the administration might be the result of environmental and/ornutritional exposure of test agents.

EXAMPLES Example 1 Cell Culture and Lysis for Mass SpectrometricExperiments

Cells were cultured in DMEM medium or RPMI 1640 medium supplemented with10% fetal bovine serum and penicillin/streptomycin. Cells were harvestedat about 80% confluency. After complete aspiration of medium from theplates, cells were scraped off the plate in 10 ml lysis buffer per 2×10⁸cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplementedwith 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate) andsonicated. Sonicated cell lysates were cleared by centrifugation at20,000×g.

Example 2 Protein Digestion

Lysate proteins were reduced with DTT at a final concentration of 4.1 mMand alkylated with iodoacetamide at 8.3 mM. For digestion, proteinextracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of2 M urea and endoproteinase GluC (Worthington, 1 mg/ml in 20 mM Hepes pH8.0) was added at 1/100 of the digestion volume. Digestion was performedovernight at room temperature.

Example 3 Reversed-phase Solid-phase Extraction of Digests

Trifluoroacetic acid (TFA) was added to protein digests to a finalconcentration of 1%, precipitate was removed by centrifugation, anddigests were loaded onto Sep-Pak C₁₈ columns (Waters) equilibrated with0.1% TFA. A column volume of 0.7-1.0 ml was used per 2×10⁸ cells.Columns were washed with 15 volumes of 0.1% TFA. A peptide fraction wasobtained by elution with 3×2 volumes of 40% MeCN in 0.1% TFA. Peptidewere lyophilized.

Example 4 Immunoaffinity Purification (IAP) of Peptides

Peptides (0.8 to 5 mg per cell sample) were dissolved in 1.4 ml of IAPbuffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl), brieflysonicated in a water bath, and insoluble matter was removed bycentrifugation. The peptide solution was added to mutation-specificantibody or mixtures of mutation-specific antibodies, with each antibodycoupled at 5 ug to a total volume of 40 ul beads of protein A agarose(Roche). The antibody/peptide mixture was to incubated overnight at 4°C. with gentle shaking. The immobilized antibody beads were washed threetimes with 1 ml IAP buffer and twice with 1 ml water, all at 0-4° C.Peptides were eluted from beads by incubation with 55 μl of 0.15% TFA atroom temperature for 10 min (eluate 1), followed by a wash of the beads(eluate 2) with 45 μl of 0.15% TFA. Both eluates were combined. The IAPeluate was purified by 0.2 μl C18 microtips (StageTips). Peptides wereeluted from the microcolumns with 4 μl of 60% MeCN, 0.1% TFA and driedin a SpeedVac. After drying, the peptide was redissolved in 5 ul of 5%MeCN, 0.1% TFA.

Example 5 Analysis by LC-MS

4 ul of redissolved peptide was loaded onto a 10 cm×75 μm PicoFritcapillary column (New Objective) packed with Magic C18 AQreversed-phase-resin (Michrom Bioresources) using a Famos autosamplerwith an inert sample injection valve (Dionex). The column was thendeveloped with a 45-min linear gradient of acetonitrile delivered at 200nl/min (Ultimate, Dionex), and MS1 spectra were collected with anOrbitrap mass spectrometer. MS1 peaks were analyzed against theoreticalmasses using MZmine software, see Katajamaa et al., Bioinformatics. 2006January; 22(5):634-6.

Example 6

Diverse cell lines each carrying one of three mutations to be detectedwith the methods described herein were harvested, and proteins weredigested with the endoproteinase GluC. After purification by C18chromatography, cellular peptides were subjected to immunoaffinitypurification (IAP) with appropriate antibodies in order to enrich forpeptides carrying the mutation screened for, and subsequent massspectrometric analysis. Peptide IAPs were performed on 5 mg of peptide.

Example 7

The mutation L858R in the EGF receptor was detected in H3255 cells afterIAP enrichment of peptide with an antibody recognizing the mutatedpeptide sequence (i.e., the mutant signature peptide). The lack ofdetection of the mutated peptide sequence in H1650 and HCC827 cells,which both do not carry this mutation, served as negative control.

Example 8

The mutation resulting in the E746-A750 deletion in the EGF receptor wasdetected in H1650 and HCC827 cells after IAP enrichment of peptide withan antibody recognizing the mutated peptide sequence. The lack ofdetection of the mutated peptide sequence in H3255 and H1975 cells,which both do not carry this mutation, served as negative control.

Example 9

The mutation G12S in the small G protein K-Ras, as well as wild-typeK-Ras, was detected in A549 cells after IAP enrichment of peptide withan antibody recognizing the peptide sequence stretching across themutated amino acid.

Example 10

Peptides from three cell lines carrying three different mutations weremixed and mutated peptides were captured with a mixture threeantibodies. Peptides from H3255 cells (for EGFR L858R mutation), H 1650cells (for EGFR del 746-750 mutation), and A 549 cells (for K-Ras G12Smutation) were mixed in equal amounts, at 0.8 mg peptide of each celllysate. The peptide mix was subjected to IAP with a mix of the threemutation antibodies. All three mutations were detected by MS1 with goodintensity. The chromatographic retention time of the peptides perfectlyaligned with the retention time of synthetic heavy-isotope peptides ofthe same amino acid sequence (i.e., standard peptides), furtherconfirming identity, beyond the accurate mass readout. Intensities ofmutated peptides were in the range of 6E5 to 8E6, while the currentdetection limit of the mass spectrometer is in the low E4 range: Thiswill allow for significant reduction in the sample amount needed fordetection.

FIG. 1 shows the co-elution of synthetic heavy isotope peptides andbiological peptides as described in this example. FIG. 2 shows theco-elution of synthetic heavy isotope peptides and biological peptideswith the coelution of 1C13 and 2C13 peptides (peptide isotopescontaining 1 and 2 C13 atoms, respectively). This coelution of peptideisotopes confirms the status of the peaks as peptides as opposed tonoise (important for lower intensities than observed in thisexperiment), and shows agreement of observed charge state of the peptidewith the theoretically expected one (if peptides were of a differentcharge state, the m/z values of the isotopes would show a differentspacing from one another). The calculated and measured m/z values of thepeptides show in FIG. 2 are set forth below in Table 1.

TABLE 1 Calculated Peptide Labeling m/z Measured m/z EGFR 737-758del746-750, 0C13 465.04456 465.04443 synth. heavy isotope peptide 1C13465.29528 465.29492 2C13 465.54597 465.54558 EGFR 737-758 del746-750,0C13 463.54111 463.54103 biological peptide 1C13 463.79183 463.791752C13 464.04252 464.04245 EGFR 830-868 L858R, synth. 0C13 748.59511748.59552 heavy isotope peptide 1C13 748.76225 748.76257 2C13 748.92937748.92957 EGFR 830-868 L858R, 0C13 747.42559 747.42609 biologicalpeptide 1C13 747.59272 747.59302 2C13 747.75984 747.76007 K-Ras 4-31G12S, synth. 0C13 748.66911 748.66956 heavy isotope peptide 1C13748.91983 748.92010 2C13 749.17054 749.17078 K-Ras 4-31 G12S, biological0C13 747.16565 747.16608 peptide 1C13 747.41638 747.41681 2C13 747.66708747.66748

Unless defined otherwise, all technical and scientific terms and anyacronyms used herein have the same meanings as commonly understood byone of ordinary skill in the art in the field of this invention.Although any compositions, methods, kits, and means for communicatinginformation similar or equivalent to those described herein can be usedto practice this invention, the preferred compositions, methods, kits,and means for communicating information are described herein.

All references cited above are incorporated herein by reference in theirentirety to the extent allowed by law. The discussion of thosereferences is intended merely to summarize the assertions made by theirauthors. No admission is made that any reference (or a portion of anyreference) is relevant prior art. Applicants reserve the right tochallenge the accuracy and pertinence of any cited reference.

What is claimed is:
 1. A kit for determining the presence of at leastone distinct polypeptide, in a biological sample, comprising a substratecomprising at least one immobilized binding partner, wherein the atleast one immobilized binding partner is capable of specifically bindinga distinct peptide containing a mutation or post-translationalmodification relative to a wild type or unmodified polypeptide, whereinthe distinct polypeptide has been hydrolyzed in a sequence specificmanner by a hydrolyzing agent into at least one distinct peptide andwherein the at least one distinct peptide has a predetermined peptidemeasured accurate mass.
 2. The kit of claim 1, further comprising atleast one standard peptide, wherein the at least one standard peptidehas substantially the same amino acid sequence as the at least onedistinct peptide but a different peptide measured accurate mass.
 3. Thekit of claim 1, further comprising at least two reference peptides,wherein the at least two reference peptides have a known liquidchromatography elution order.
 4. The kit of claim 2, wherein thestandard peptide comprises an isotope selected from the group consistingof ¹⁵N, ¹³C, ¹⁸O and ²H.
 5. The kit of claim 2, wherein the at least onestandard peptide and the at least one distinct peptide co-segregateduring liquid chromatography.
 6. The kit of claim 2, wherein the atleast one standard peptide and the at least one distinct peptide have asubstantially identical retention time.
 7. The kit of claim 1, whereinthe mutation is an addition, deletion and/or substitution of about 1 toabout 10 amino acid residues.
 8. The kit of claim 1, wherein thepost-translational modification is selected from the group consisting ofphosphorylation, acetylation, ubiquitination, and glycosylation.
 9. Thekit of claim 1, wherein the kit further comprises the hydrolyzing agent.10. The kit of claim 9, wherein the hydrolyzing agent is selected fromthe group consisting of an enzyme and a chemical.
 11. The kit of claim9, wherein the hydrolyzing agent is selected from the group consistingof cyanogen bromide, BNPS-Skatole, formic acid, trypsin, Lysine-Cendopeptidase (LysC); arginine-C endopeptidase (ArgC), Asp-N, glutamicacid, endopeptidase (GluC), chymotrypsin, and combinations thereof. 12.The kit of claim 1, wherein the substrate comprises a substance selectedfrom the group consisting of a gel matrix and polymer beads.
 13. The kitof claim 1, wherein the at least one immobilized binding partnercomprises a reagent selected from the group consisting of a protein thatspecifically binds to the distinct peptide, and an antibody or antibodyfragment that specifically binds a mutant signature peptide.
 14. The kitof claim 1, wherein the at least one immobilized binding partner is 1 toabout 500 immobilized binding partners, wherein each immobilized bindingpartner specifically binds to a different distinct peptide.
 15. The kitof claim 1, wherein the at least one immobilized binding partner is atleast 500 immobilized binding partners, wherein each immobilized bindingpartner specifically binds to a different distinct peptide.
 16. The kitof claim 1, wherein the kit further comprises an elution solutioncapable of causing the dissociation of the distinct peptide from theimmobilized binding partner into the elution solution.